Structure of Hydroxyl Radical-induced DNA-protein Crosslinks in Calf Thymus Nucleohistonein Vitro

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Abstract
Hydroxyl radicals are known to produce DNA—protein crosslinks in chromatin in vivo and in vitro. Here we investigated DNA—protein crosslinks formed between aliphatic amino acids and thymine in calf thymus nucleohistone exposed predominantly to hydroxyl radicals in γ-irradiated N2O-saturated aqueous solution. Aliphatic amino acids are the predominant types of amino acids in the core histones of calf thymus, and thus are likely to form crosslinks with DNA. For identification of the crosslinks we first investigated hydroxyl radical-induced crosslinking of thymine to aliphatic amino acids in model systems, i.e. an aqueous mixture of thymine and a single amino acid. Samples were analyzed for possible thymine—amino acid crosslinks by gas chromatography—mass spectrometry. Using this approach the structure of the crosslinks was elucidated, and information on their gas chromatographic and mass spectral properties was obtained. Gas chromatography—mass spectrometry with selected-ion monitoring (GC-MS/SIM) was then used to identify DNA—protein crosslinks in acidic hydrolysates of calf thymus nucleohistone exposed to ionizing radiation in buffered aqueous solution. DNA—protein crosslinks involving thymine and the amino acids Gly, Ala, Val, Leu, Ile and Thr were identified. In some cases several isomers of the same crosslink were observed. The yield of the crosslinks was measured by GC-MS/SIM and was found to be a linear function of radiation dose in the range 49 to 436 Gy. The mechanism for the formation of these DNA—protein crosslinks is thought to involve hydrogen atom abstraction by hydroxyl radicals from the aliphatic amino acid followed by addition of the amino acid radical to the carbon(6)-position of thymine and subsequent oxidation of the adduct radical.